Biomedical subjects

M D Waterfield

Publications and source records attributed to M D Waterfield.

At least 19 recordsLinked to original sources

PubMed1995

Modulation of the substrate specificity of the mammalian phosphatidylinositol 3-kinase by cholesterol sulfate and sulfatide.

The substrate specificity of the purified, mammalian phosphatidylinositol 3-kinase is subject to modulation by detergents, which are able to switch substrate specificity in vitro in favor of PtdInsP2. This effect of the detergents is due to an activation of the phosphatidylinositol biphosphate 3-kinase activity, while the phosphatidylinositol 3-kinase activity is inhibited. The selective inhibition of the phosphatidylinositol 3-kinase activity (p110 alpha/p85 alpha) is shown here also to be observed by employing cholesterol sulfate or sulfatide at low micromolar concentrations, whereas cholesterol and androsterone sulfate fail to inhibit. These naturally occurring sulfated lipids have at these concentrations no effect on the phosphatidylinositol bisphosphate 3-kinase activity but inhibit the manganese-dependent intrinsic protein kinase activity, thus switching substrate specificity toward the more highly phosphorylated inositol lipids. Cholesterol sulfate and sulfatide inhibit the free catalytic subunit p110 alpha but fail to inhibit the homologous phosphatidylinositol 3-kinase from Saccharomyces cerevisiae (Vps34p), suggesting that these sulfated lipids act specifically on the mammalian phosphatidylinositol 3-kinase. Consistent with this specificity, the regulatory subunit (p85), which is not conserved in the yeast enzyme, is found to play an important role for the affinity of these inhibitors. The implications for the phosphatidylinositol 3-kinase activity in vivo are discussed.

Adenosine Triphosphate↗

PubMed1995

A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system.

Phosphoinositide (PI) 3-kinases have been characterized as enzymes involved in receptor signal transduction in mammalian cells and in a complex which mediates protein trafficking in yeast. PI 3-kinases linked to receptors with intrinsic or associated tyrosine kinase activity are heterodimeric proteins, consisting of p85 adaptor and p110 catalytic subunits, which can generate the 3-phosphorylated forms of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 as potential second messengers. Yeast Vps34p kinase, however, has a substrate specificity restricted to PtdIns and is a PtdIns 3-kinase. Here the molecular characterization of a new human PtdIns 3-kinase with extensive sequence homology to Vps34p is described. PtdIns 3-kinase does not associate with p85 and phosphorylates PtdIns, but not PtdIns4P or PtdIns(4,5)P2. In vivo PtdIns 3-kinase is in a complex with a cellular protein of 150 kDa, as detected by immunoprecipitation from human cells. Protein sequence analysis and cDNA cloning show that this 150 kDa protein is highly homologous to Vps15p, a 160 kDa protein serine/threonine kinase associated with yeast Vps34p. These results suggest that the major components of the yeast Vps intracellular trafficking complex are conserved in humans.

Amino Acid Sequence↗

PubMed1995

Normal activation of p70 S6 kinase by insulin in cells overexpressing dominant negative 85kD subunit of phosphoinositide 3-kinase.

The role of heteromeric phosphoinositide (PI) 3-kinase activity in insulin signal transduction was studied by investigating the effects of (i) overexpression of a dominant negative mutant p85 (delta p85) that lacks the binding site for p110 (delta p85-overexpressing cells) and (ii) inhibition of PI 3-kinase activity by wortmannin (wortmannin-treated cells). The insulin-induced association of PI 3-kinase activity with insulin receptor sustrate-1 (IRS-1) was inhibited in both wortmannin-treated cells and delta p85-overexpressing cells. However, whereas insulin-induced activation of p70 S6 kinase was completely abolished in wortmannin-treated cells, it appeared normal in delta p85-overexpressing cells. These results raise the possibility that a wortmannin-sensitive pathway independent of heteromeric PI 3-kinase is involved in the activation of p70 S6 kinase by insulin.

Amino Acid Sequence↗

PubMed1994

Modulation of Kit/stem cell factor receptor-induced signaling by protein kinase C.

The Kit/stem cell factor receptor (Kit/SCF-R) is a transmembrane tyrosine kinase receptor of importance for the normal development of hemopoietic cells, melanoblasts, and germ cells. We recently reported that protein kinase C (PKC) is involved in a negative feedback loop regulating the Kit/SCF-R by direct phosphorylation on serine residues in the receptor. Inhibition of PKC led to increased SCF-induced tyrosine kinase activity and mitogenicity, but PKC was necessary for SCF-induced motility. In this report we have further examined the modulatory role of PKC on SCF-induced signaling. The ligand-activated Kit/SCF-R associated weakly with GRB2 and induced only little tyrosine phosphorylation of phospholipase C-gamma in porcine aortic endothelial cells transfected with Kit/SCF-R. In contrast, the SCF-stimulated Kit/SCF-R associated efficiently with, and induced tyrosine phosphorylation of, the p85 alpha regulatory subunit of phosphatidyl inositide-3'-kinase (PI-3'-kinase). Both receptor association and tyrosine phosphorylation of p85 alpha were increased after inhibition of PKC, while its serine phosphorylation was decreased. Concomitantly, the specific activity of receptor-associated PI-3'-kinase activity was increased. Inhibition of PI-3'-kinase with wortmannin inhibited SCF-induced mitogenicity. SCF-induced phosphorylation of Raf-1 and activation of ERK2 still occurred after PKC inhibition but was not increased. In conclusion, SCF-induced PI-3'-kinase activation paralleled the increased SCF-induced mitogenicity after inhibition of PKC.

Adaptor Proteins, Signal Transducing↗

PubMed1994

The catalytic subunit of phosphatidylinositol 3-kinase is a substrate for the activated platelet-derived growth factor receptor, but not for middle-T antigen-pp60c-src complexes.

The interaction of phosphatidylinositol 3-kinase (PI 3-K) with polyoma-virus middle-T antigen-pp60c-src (mT:cSrc) complexes and with the platelet-derived growth factor (PDGF) receptor has been investigated. Firstly, we undertook reconstitution studies, using proteins derived from a baculovirus expression system. The p110 catalytic subunit of the PI 3-K associated with tyrosine kinases only when complexed with the p85 alpha regulatory subunit. Both p85 alpha and p110 were substrates of the PDGF receptor. In contrast, only the p85 alpha subunit was detectably phosphorylated when PI 3-K was associated with mT:cSrc. Secondly, we studied PI 3-K in mammalian cells. In mT-antigen-transformed NIH-3T3 cells neither p85 alpha nor p110 was phosphorylated on tyrosine residues in vivo, even though p85 alpha was a substrate in kinase assays in vitro. In quiescent NIH-3T3 cells, PI 3-K showed detectable activity in vitro; PDGF stimulation resulted in a rapid and transient association of PI 3-K with the receptor, which was correlated with a transient increase in intrinsic P13-K activity (approx. 2-fold). The activated PDGF receptor phosphorylated p110 in vitro, at one major site. In vivo, PDGF stimulation induced tyrosine phosphorylation of p110 that persisted for at least 1 h after stimulation. Immunodepletion of the PDGF receptor from stimulated cell lysates showed that p110 was released from the receptor in a tyrosine-phosphorylated form. From these results we conclude that (i) the mT:cSrc complex and the PDGF receptor differ in their association with PI 3-K activity, (ii) PDGF receptor appears to activate PI 3-K in vivo both by relocation of the enzyme and by stimulation of its intrinsic activity, and (iii) tyrosine phosphorylation of the p110 subunit by the PDGF receptor may play a role in PI 3-K regulation in some circumstances.

3T3 Cells↗

PubMed1994

Molecular cloning, cDNA sequence, and chromosomal localization of the human phosphatidylinositol 3-kinase p110 alpha (PIK3CA) gene.

Phosphatidylinositol (PI) 3-kinase is a heterodimeric enzyme comprising a 110-kDa catalytic subunit and an 85-kDa regulatory subunit that binds to tyrosine phosphopeptide sites linked directly or indirectly to receptors serving diverse signal functions. Knowledge of the structure and function of PI 3-kinase was greatly advanced by the purification, cDNA cloning, and subsequent expression of the bovine enzyme. Here the cloning of the cDNA for the human p110 alpha subunit of PI 3-kinase (PIK3CA), encoding a protein 99% identical to the bovine p110, and of its gene in YAC is described. The chromosomal localization of the gene for PIK3CA is shown to be at 3q21-qter as determined using somatic cell hybrids. In situ hybridization performed using Alu-PCR from the YAC DNA located the gene in 3q26.3.

Animals↗

PubMed1993

Association of the p85 regulatory subunit of phosphatidylinositol 3-kinase with an essential erythropoietin receptor subdomain.

Using an active, HAI epitope-tagged form of the murine erythropoietin (EPO) receptor and via direct coimmunoprecipitation, the p85 regulatory subunit of phosphatidyl inositol-3 kinase (p85/PI3-K) is shown to associate with the EPO receptor in transfected FDC-P1 cell lines. Coimmunoprecipitation of p85 with epitope-tagged EPO receptors was observed initially in FDC-HER cells labeled metabolically with [32P]orthophosphate, and association of these factors was confirmed by Western analyses of receptor immunoprecipitates using p85 antiserum. Interestingly, this association occurred in the absence of ligand, and exposure of FDC-HER cells to EPO did not detectably affect levels of receptor-associated p85 or overall levels of p85 phosphorylation. However, EPO was observed to stimulated the rapid formation of phosphatidylinositol 32P-phosphate in FDC-HER and FDC-ER cells. Through baculovirus-mediated expression of epitope-tagged EPO receptor forms in SF9 cells, domains for p85 association were mapped. Analyses of receptor forms with cytosolic truncations and deletions delineated a candidate subdomain for p85 binding to an essential extended box-2 region (P329-E374; including a putative motif for SH2 binding, Y343LVL). These findings extend a mechanistic alignment between the EPO receptor and protein tyrosine kinase-encoding receptors that likewise activate PI3-K, and expand the importance of further defining pathways to PI3-K activation.

Amino Acid Sequence↗

PubMed1993

Solution structure and ligand-binding site of the SH3 domain of the p85 alpha subunit of phosphatidylinositol 3-kinase.

SH3 domains are found in proteins associated with receptor tyrosine kinase signal transduction complexes. The solution structure of the SH3 domain of the 85 kd regulatory subunit of phosphatidylinositol 3-kinase is shown to be a compact beta barrel consisting of five beta strands arranged in two beta sheets of three and two strands. The structure is similar to that of chicken brain alpha spectrin but represents a distinct class of SH3 domain, with an insertion between the second and third beta strands that may influence binding specificity. 1H chemical shift changes induced by complex formation with a synthetic peptide derived from the SH3-binding protein dynamin, together with amino acid sequence comparisons, suggest that the ligand-binding site consists of a hydrophobic surface flanked by two charged loops.

Amino Acid Sequence↗

PubMed1993

A biosensor approach to probe the structure and function of the p85 alpha subunit of the phosphatidylinositol 3-kinase complex.

Phosphatidylinositol 3-kinase, which generates putative novel second messenger phospholipids, is a heterodimer composed of regulatory adaptor 85-kDa and catalytic 110-kDa subunits. The p85 alpha subunit contains a NH2-terminal src homology (SH) 3 domain, a region with homology to the product of the breakpoint cluster region (bcr) gene, and a COOH-terminal portion of the molecule which contains two SH2 domains, separated by a spacer region. In this study a panel of monoclonal antibodies (mAb) was raised against recombinant bovine p85 alpha to probe its multidomain structure in relation to function. These mAbs were characterized using a BIAcore biosensor instrument. Epitopes for nine mAbs were mapped in relation to the domain structure of p85 alpha using recombinant protein fragments expressed in bacteria. These mAbs were then used to map the sites on p85 alpha which are involved in growth factor receptor binding. Two interesting classes of functional mAbs were identified. First, mAb U14, whose epitope lies within the NH2-terminal SH2 domain of p85 alpha, blocked the interaction of p85 alpha with activated protein-tyrosine kinase receptors. Second, real-time binding experiments using phospholipid-containing vesicles showed that p85 alpha by itself could specifically bind certain phospholipids. Two mAbs (U9 and U15) with epitopes located in the inter-SH2 spacer region blocked the binding of lipids to this site. The relevance of these observations to understanding the relationship of structure to function of p85 and the phosphatidylinositol 3-kinase are discussed.

Amino Acid Sequence↗

PubMed1993

Activation of Src family kinases by colony stimulating factor-1, and their association with its receptor.

The receptor for the macrophage colony stimulating factor-1 (CSF-1R) is a transmembrane glycoprotein with intrinsic tyrosine kinase activity. CSF-1 stimulation promotes the growth of cells of the macrophage lineage and of fibroblasts engineered to express CSF-1R. We show that CSF-1 stimulation resulted in activation of three Src family kinases, Src, Fyn and Yes. Concomitant with their activation, all three Src family kinases were found to associate with the ligand-activated CSF-1 receptor. These interactions were also demonstrated in SF9 insect cells co-infected with viruses encoding the CSF-1 receptor and Fyn, and the isolated SH2 domain of Fyn was capable of binding the CSF-1R in vitro. Analysis of mutant CSF-1Rs revealed that the 'kinase insert' (KI) domain of CSF-1R was not required for interactions with Src family kinases, but that mutation of one of the receptor autophosphorylation sites, Tyr809, reduced both their binding and enzymatic activation. Because fibroblasts expressing this receptor mutant are unable to form colonies in semi-solid medium or to grow in chemically defined medium in the presence of CSF-1, the Src family kinases may play a physiological role in the mitogenic response to CSF-1.

3T3 Cells↗

PubMed1993

A novel recognition motif for phosphatidylinositol 3-kinase binding mediates its association with the hepatocyte growth factor/scatter factor receptor.

The pleiotropic effects (mitogenesis, motogenesis, and morphogenesis) elicited by hepatocyte growth factor/scatter factor (HGF/SF) are mediated by the activation of the tyrosine kinase receptor encoded by the MET proto-oncogene. Following autophosphorylation, the receptor associates with the p85/110 phosphatidylinositol (PI) 3-kinase complex in vivo and in vitro. By a combination of two complementary approaches, competition with synthetic phosphopeptides and association with Tyr-Phe receptor mutants, we have identified Y-1349 and Y-1356 in the HGF/SF receptor as the binding sites for PI 3-kinase. Y-1349VHV and Y-1356VNV do not conform to the canonical consensus sequence YXXM for PI 3-kinase binding and thus define YVXV as a novel recognition motif. Y-1349 and Y-1356 are located within the C-terminal portion of the HGF/SF receptor and are phosphorylation sites. The affinity of the N- and C-terminal src homology region 2 (SH2) domains of p85 for the phosphopeptides including Y-1349 and Y-1356 is 2 orders of magnitude lower than that measured for Y-751 in the platelet-derived growth factor receptor binding site. However, the closely spaced duplication of the novel recognition motif in the native HGF/SF receptor may allow binding with both SH2 domains of p85, thus generating an efficient docking site for PI 3-kinase. In agreement with this model, we have observed that a phosphopeptide including both Y-1349 and Y-1356 activates PI 3-kinase in vitro.

Amino Acid Sequence↗

PubMed1992

Purification and characterization of a phosphatidylinositol 3-kinase complex from bovine brain by using phosphopeptide affinity columns.

Specific phosphorylated tyrosine residues in the kinase insert region of the human platelet-derived-growth-factor beta-receptor mediate the formation of multienzyme complexes with this receptor. When phosphorylated, tyrosine residue 751 within the kinase insert region mediates binding of PtdIns 3-kinase to this receptor. A 17-amino-acid peptide containing this tyrosine residue was synthesized, phosphorylated by using epidermal-growth-factor receptor and then coupled to an Actigel matrix. The tyrosine-751 phosphopeptide column is used here as a final affinity step in the purification of the PtdIns 3-kinase from bovine brain to apparent homogeneity. The active resin-bound PtdIns 3-kinase is composed of two polypeptides, p110 and p85, which are elutable with SDS-containing buffers and detectable by silver staining of polyacrylamide gels. The 85 kDa protein is shown to be identical with the recently cloned p85 alpha. Phosphotyrosine is demonstrated to be an essential part of the structure required for binding of both of these proteins and PtdIns 3-kinase activity to this peptide. The active PtdIns 3-kinase complex from bovine brain, but not recombinant p85 subunits, shows specificity for binding to phosphopeptides containing a YXXM consensus sequence. Neither PtdIns 3-kinase activity, nor the complex of p85 and 110 kDa proteins, binds to several other phosphopeptide affinity columns lacking this sequence motif. The selectivity of binding of baculovirus-expressed free p85 alpha subunit of bovine brain PtdIns 3-kinase, the closely related protein p85 beta and purified bovine brain PtdIns 3-kinase to these and other phosphopeptide columns is examined.

Amino Acid Sequence↗

PubMed1992

Expression and characterization of the p85 subunit of the phosphatidylinositol 3-kinase complex and a related p85 beta protein by using the baculovirus expression system.

PtdIns 3-kinase associates with certain activated protein-tyrosine kinase receptors and with the pp60c-src/polyoma middle-T complex, suggesting that the enzyme is involved in growth regulation. The purified PtdIns 3-kinase appears to have two subunits, of 85 kDa and 110 kDa. Structural analysis at protein and cDNA levels revealed two forms of the 85 kDa subunit, one which associates with PtdIns 3-kinase activity termed p85 alpha, and a protein of unknown function, p85 beta. Both 85 kDa proteins contain src-homology regions 2 and 3 (SH2 and SH3), but lack enzymic activity, suggesting that they may be regulatory subunits of PtdIns 3-kinase. To probe their structure and function further, p85 alpha and p85 beta have been expressed and purified in large amounts from insect cells by using baculovirus vectors. Specific antisera detect p85 alpha, but not p85 beta, associated with PtdIns 3-kinase activity in various cell types. Co-expression studies in insect cells have shown that p85 alpha and p85 beta are substrates for the protein-tyrosine kinases of epidermal growth factor, colony-stimulating factor 1 and c-erbB2 receptors and the src family kinase p59c-fyn. Both p85 alpha and p85 beta form tight complexes with these protein-tyrosine kinases as measured by immunoprecipitation and kinase assays in vitro. The specificity of binding of free p85 is less restricted than that of p85 in the active PtdIns 3-kinase complex with the 110 kDa protein. The relevance of these results to growth-factor-induced PtdIns 3-kinase activation is discussed.

Amino Acid Sequence↗

PubMed1992

Regulation of phosphoinositide kinases in T cells. Evidence that phosphatidylinositol 3-kinase is not a substrate for T cell antigen receptor-regulated tyrosine kinases.

A phosphoinositide kinase that can phosphorylate phosphatidylinositol (PtdIns) is present in 4G10 monoclonal antibody (mAb) phosphotyrosine immunoprecipitates isolated from T cells activated via the T cell antigen receptor (TCR).CD3 complex. This PtdIns kinase is not the PtdIns 3-kinase that associates with activated protein tyrosine kinases in fibroblasts, since Western blotting and immunoprecipitation experiments with antibodies specific for the p85 alpha subunit of the PtdIns 3-kinase indicate that this polypeptide is not immunoprecipitated by the 4G10 mAb from TCR.CD3-activated Jurkat cells. Moreover, immunoprecipitated PtdIns 3-kinase isolated from T cells with p85 antibodies is inhibited when PtdIns is presented in Nonidet P-40, whereas the PtdIns kinase activity present in 4G10 mAb phosphotyrosine immunoprecipitates is enhanced in the presence of Nonidet P-40. In vitro kinase assays of PtdIns 3-kinase immunoprecipitated with p85 antibodies from T cells indicate that it associates with a serine kinase that can phosphorylate a p85 polypeptide. However, no protein tyrosine kinase activity capable of tyrosine phosphorylating p85 in vitro associates with p85 alpha immunoprecipitates in quiescent or TCR.CD3-activated T cells. These data suggest that the TCR.CD3 complex does not regulate PtdIns 3-kinase activity by a mechanism that involves protein tyrosine kinases.

Adenosine↗

PubMed1992

Tenascin: a modulator of cell growth.

The large, multidomain extracellular matrix protein tenascin displays a markedly restricted tissue distribution during embryogenesis and remains present only in a few adult tissues. The protein is reexpressed, however, during wound healing and in the stroma of malignant tumours. While a variety of studies have dealt with the important role of tenascin in the development of neural and non-neural tissues, there is growing evidence that tenascin expression may be associated with proliferation of cells lining these tissues. The presence of repeating domains in tenascin similar to those in epidermal growth factor prompted us to investigate the ability of tenascin to modulate the growth of different cell types. Tenascin was actually found to be mitogenic for several cell types. This mitogenic activity, however, appears to be associated with a region in the fibronectin type III domains. The mitogenic mechanism is clearly distinct from pathways used by peptide growth factors such as epidermal growth factor and platelet-derived growth factor, which activate the intrinsic tyrosine kinase activity of their cell-surface receptors. However, we show that this large extracellular matrix molecule is efficiently internalised and may be processed by responding cells.

3T3 Cells↗

PubMed1992

Structure of an SH2 domain of the p85 alpha subunit of phosphatidylinositol-3-OH kinase.

Receptor protein-tyrosine kinases, through phosphorylation of specific tyrosine residues, generate high-affinity binding sites which direct assembly of multienzyme signalling complexes. Many of these signalling proteins, including phospholipase C gamma, GTPase-activating protein and phosphatidylinositol-3-OH kinase, contain src-homology 2 (SH2) domains, which bind with high affinity and specificity to tyrosine-phosphorylated sequences. The critical role played by SH2 domains in signalling has been highlighted by recent studies showing that mutation of specific phosphorylation sites on the platelet-derived growth factor receptor impair its association with phosphatidylinositol-3-OH kinase, preventing growth factor-induced mitogenesis. Here we report the solution structure of an isolated SH2 domain from the 85K regulatory subunit of phosphatidylinositol-3-OH kinase, determined using multidimensional nuclear magnetic resonance spectroscopy. The structure is characterized by a central region of beta-sheet flanked by two alpha-helices, with a highly flexible loop close to functionally important residues previously identified by site-directed mutagenesis.

Animals↗

PubMed1992

Activated phosphoinositide 3-kinase associates with membrane skeleton in thrombin-exposed platelets.

Human platelets undergo a rapid, major reorganization of the cytoskeletal matrix upon exposure to thrombin, and accumulate 3-phosphorylated phosphoinositides in a protein kinase C (PKC)-dependent manner. These phosphoinositides have been suggested to be involved in actin polymerization/depolymerization. We reasoned that, if newly generated 3-phosphorylated phosphoinositide modulates cytoskeletal reorganization, a prerequisite for such action would be generation near cytoskeletal proteins. We have found that, after platelet activation, phosphatidylinositol 3-kinase and phosphatidylinositol(4)P 3-kinase activities, antibody-detectable phosphoinositide 3-kinase, and PKC become markedly and specifically enriched in a Triton X-100-insoluble cytoskeletal fraction that contains GPIIb/IIIa (integrin) and pp60c-src. The cytoskeletal fraction then accounts for up to 70% of total phosphoinositide 3-kinase activity, a function of recruited activated enzyme. These proteins are not occluded or directly associated with newly polymerized actin, since blockage by cytochalasin D of actin polymerization, and consequent inhibition of accumulation of about 40% of incremental protein and actin in this fraction, has no effect on its content of phosphoinositide 3-kinase, GPIIb/IIIa, pp60c-src, or PKC. Depolymerization of actin with DNase I, or inhibition of ligand binding to GPIIb/IIIa by RGDS, however, in combination with cytochalasin D, further depletes actin and significantly decreases sedimentability of GPIIb/IIIa as well as phosphoinositide 3-kinase, pp60c-src, and PKC, without inhibiting total 3-kinase activity. Our results suggest that, as a function of platelet activation, enzymes that regulate the synthesis of 3-phosphorylated phosphoinositides rapidly associate with the membrane skeleton and that skeletally associated phosphoinositide 3-kinase is more active than the Triton-soluble form.

1-Phosphatidylinositol 4-Kinase↗

PubMed1992

Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha.

Amino Acids↗